Cardiovascular diseases are the major cause of mortality worldwide. Regeneration of the damaged myocardium remains a challenge due to mechanical constraints and limited healing ability of the adult heart tissue. Cardiac tissue engineering using biomaterial scaffolds combined with stem cells and bioactive molecules could be a highly promising approach for cardiac repair. Use of biomaterials can provide suitable microenvironment to the cells and can solve cell engraftment problems associated with cell transplantation alone. Mesenchymal stem cells (MSCs) are potential candidates in cardiac tissue engineering because of their multilineage differentiation potential and ease of isolation. Use of DNA methyl transferase inhibitor, such as zebularine, in combination with three-dimensional (3D) scaffold can promote efficient MSC differentiation into cardiac lineage, as epigenetic modifications play a fundamental role in determining cell fate and lineage specific gene expression.

To investigate the role of collagen scaffold and zebularine in the differentiation of rat bone marrow (BM)-MSCs and their subsequent in vivo effects.

MSCs were isolated from rat BM and characterized morphologically, immunophenotypically and by multilineage differentiation potential. MSCs were seeded in collagen scaffold and treated with 3 μmol/L zebularine in three different ways. Cytotoxicity analysis was done and cardiac differentiation was analyzed at the gene and protein levels. Treated and untreated MSC-seeded scaffolds were transplanted in the rat myocardial infarction (MI) model and cardiac function was assessed by echocardiography. Cell tracking was performed by DiI dye labeling, while regeneration and neovascularization were evaluated by histological and immunohistochemical analysis, res pectively.

MSCs were successfully isolated and seeded in collagen scaffold. Cytotoxicity analysis revealed that zebularine was not cytotoxic in any of the treatment groups. Cardiac differentiation analysis showed more pronounced results in the type 3 treatment group which was subsequently chosen for the transplantation in the in vivo MI model. Significant improvement in cardiac function was observed in the zebularine treated MSC-seeded scaffold group as compared to the MI control. Histological analysis also showed reduction in fibrotic scar, improvement in left ventricular wall thickness and preservation of ventricular remodeling in the zebularine treated MSC-seeded scaffold group. Immunohistochemical analysis revealed significant expression of cardiac proteins in DiI labeled transplanted cells and a significant increase in the number of blood vessels in the zebularine treated MSC-seeded collagen scaffold transplanted group.

Combination of 3D collagen scaffold and zebularine treatment enhances cardiac differentiation potential of MSCs, improves cell engraftment at the infarcted region, reduces infarct size and improves cardiac function.

Differentiation potency of human dental pulp cells (hDPCs) is essential for dentin regeneration. DNA methylation is one of the major epigenetic mechanisms and is suggested to involve in differentiation of hDPCs, the machinery of which includes DNA methyltransferase enzymes (DNMTs) and methyl-CpG-binding domain proteins (MBDs). Our previous study has found that melatonin (MT) promoted hDPC differentiation, but its mechanism remains elusive. We aimed to investigate the role of DNA methylation in the promotion of MT to differentiation of hDPCs in vitro. hDPCs were cultured in basal growth medium (CO) or odontogenic medium (OM) exposed to MT at different concentrations (0, 10-12, 10-10, 10-8, 10-6, 10-4 M). The cell growth was analyzed using Cell Counting Kit-8 assay, and mineralized tissue formation was measured using Alizarin red staining. The expression of the 10 genes (DNMT1, DNMT3A, DNMT3B, MBD1-6, MeCP2) was determined using real-time qPCR and western blotting. The abundance of MeCP2 in the nuclei was evaluated using immunofluorescence analysis. Global methylation level was tested using ELISA. We found that mineralized tissue formation significantly increased in OM with MT at 10-4 M, while the levels of MeCP2 and global DNA methylation level declined. The expression of MBD1, MBD3, and MBD4 significantly increased in OM alone, and the expession of DNMT1 and MBD2 was decreased. These results indicate that MT promotes odontogenic differentiation of hDPCs in vitro by regulating the levels of DNMT1, MeCP2, and global DNA methylation, suggesting that MT-induced DNA methylation machinery may play an important role in tooth regeneration.